In recent years, in the field of production of medicine, gene therapy, regenerative medicine, immunotherapy etc., it is required to culture efficiently a large amount of cells, tissues, microorganisms etc. in an artificial environment.
Under such circumstances, a large amount of cells are cultured automatically in a closed system by enclosing cells and a culture medium in a culture container formed of a gas-permeable film.
In particular, when culturing lymphocytes, it is necessary to conduct culture aiming at activating lymphocytes by using anti-CD3 antibodies prior to culturing in order to increase the number of cells. Therefore, by admitting lymphocytes and a culture medium into a culture container in which anti-CD3 antibodies are contained, activation of lymphocytes is conducted.
Meanwhile, when only a small amount of anti-CD3 antibodies in the form of a solution is present in a culture container, stimulation to lymphocytes becomes insufficient and activation cannot be conducted sufficiently. On the other hand, when a large amount of anti-CD3 antibodies in the form of a solution is present in a culture container, CD3 antigens present in cell membranes are removed from by the defense mechanism of cells, whereby activation of lymphocytes is inhibited.
Immunosuppressive agents containing anti-CD3 antibodies as a main component were produced as a result of attention paid to such properties of lymphocytes, and when they are administered to patients who had rejection after organ/tissue transplantation, a large amount of anti-CD3 antibodies are bound to antigens and then accumulated, and the accumulated product is taken in the cells and discharged outside the cells. Alternatively, the accumulated product is cleaved by enzymes and antigens are removed from the cell surface. As a result, the stimulation is no longer transmitted to the inside of the cell, and the activation of lymphocytes is suppressed.
As mentioned above, it is difficult to appropriately control the activation of lymphocytes by using antibodies liberated in a solution. Conventionally, in general,—anti-CD3 antibodies are immobilized on the inner surface of the bottom of a culture container, and appropriate stimulation apply to lymphocytes by using such a culture container, thereby activation of lymphocytes has been widely conducted.
When producing a culture bag for activation of lymphocytes (hereinafter, often simply referred to as the “activation bag”), after enclosing an antibody solution containing anti-CD3 antibodies in a bag and immobilizing the anti-CD3 antibodies on the inner surface of the bag, the inside of the bag is washed to remove liberated antibodies, and thereafter, lymphocytes and a culture medium are enclosed to conduct culture for activation. By washing the inside of the bag to remove liberated antibodies, it was possible to activate lymphocytes more efficiently.
Patent Document 1: JP-A-2007-17502B
Under such circumstances, in order to examine adverse effects exerted on activation by liberated antibodies when an antibody solution remains in an activation bag, the inventors conducted activation of lymphocytes by using antibody solutions at various concentrations.
As a result, it has been found the following. When the concentration of antibodies is relatively high, i.e. as high as that used for production of an activation bag, activation of lymphocytes is suppressed. However, surprisingly, when the concentration of antibodies is low in a certain range, activation of lymphocytes is promoted.
That is, it has been revealed that, by immobilizing anti-CD3 antibodies in a bag for activation and by enclosing a low-concentration liberated antibody, it becomes possible to promote activation of lymphocytes by the activation bag, and as a result, proliferation efficiency of lymphocytes can be further improved.
Here, in Example 1 of the Patent Document 1, after an antibody solution containing anti-CD3 antibodies is enclosed within a bag and the anti-CD3 antibodies are immobilized on the inner surface of the bag, activation is conducted with the antibody solution being enclosed in order to produce an activation bag for lymphocytes. Further, in Example 2 of the Patent Document 1, after immobilizing anti-CD3 antibodies on the inner surface of the bag, the bag is washed to remove liberated antibodies, and then lymphocytes and a culture medium are enclosed to conduct activation. Patent Document 1 also states that in Example 1 in which activation is conducted with the antibody solution being enclosed, proliferation efficiency of lymphocytes was lower as compared with Example 2 in which liberated antibodies were removed by washing the inside of the bag.
That is, it is thought that, in Example 1, activation of lymphocytes was suppressed since a large amount of liberated antibodies were remained in the bag, and hence, proliferation efficiency of lymphocytes was lower than that in Example 2.
On the other hand, Patent Document 1 neither describe nor suggest that liberated antibodies in a certain low concentration range are enclosed in an activation bag in which anti-CD3 antibodies are immobilized.